PROJECT SUMMARY Adolescents account for 42% of new HIV infections worldwide, and almost 90% of these infections are acquired across the anogenital mucosa1,2. Beyond behavioral risk factors, the influences of the dramatic, dynamic shifts in hormones during adolescent reproductive development on the anal and vaginal mucosa may drive mucosal vulnerability to HIV infection. Extreme hormonal shifts in transgender adolescents undergoing cross-sex hormone therapy may contribute to the alarmingly high 25% transmission rates seen in this group1,3- 6. This proposal directly addresses adolescent biologic risk factors for HIV susceptibility in gender conforming (cis) and transgender (trans) adolescents, taking advantage of the unique hormonal manipulation in trans individuals to define the influence of testosterone and estrogen on mucosal integrity and inflammation within the anal and vaginal mucosa. Comparisons to conventional puberty in cis adolescents provide the opportunity to refine our understanding the mucosal effects of sex-steroids. We will define normative indices of anogenital microbial communities using 16s rRNA sequencing and mass spectrometry proteomics of vaginal proteins. These normative values will be evaluated in the context of blood hormone levels, Tanner sexual maturity, and mucosal trauma from self-reported sexual activity (ACASI) throughout the individual's progression either through: (1) conventional sexual maturation in cis adolescents, or (2) during pubertal hormonal blockade with gonadotropin-releasing hormone (GnRH), and subsequent sexual maturation with cross-sex hormones (estrogen, testosterone) in trans adolescents. We will obtain rectal biopsies from cis and trans gender youth, and use an ex vivo rectal tissue model to evaluate the impact of sex steroid hormones, sexual trauma and microbial communities on HIV infection. Furthermore, we will identify proteomic signatures of vaginal inflammation, mucosal barrier disruption, and differences in anogenital microbial communities that may be related to HIV susceptibility. This study will ultimately characterize the effects of sex steroid hormones and sexual trauma on commensal anogenital microbial communities and vaginal mucosal proteins that confer increase risk to mucosal HIV transmission in adolescents. This study provides desperately-needed public health data to clarify biologic risk factors that contribute to HIV acquisition and pathogenesis in these at-risk adolescent populations, in particular the effects of reproductive maturation and injury upon anogenital mucosal environments. The information gained will provide a significant platform for future hypothesis-generating studies that address modulation of HIV susceptibility and efficacious biomedical HIV prevention strategies in this highly vulnerable population.